biodiesel production.

Biotechnology Journal

Biotechnol. J. 2014, 9, 1536–1546

DOI 10.1002/biot.201400266

www.biotechnology-journal.com

Research Article

Volatile fatty acids derived from waste organics provide an economical carbon source for microbial lipids/biodiesel production Gwon Woo Park1,*, Qiang Fei1,*, Kwonsu Jung1, Ho Nam Chang1, Yeu-Chun Kim1, Nag-jong Kim2, Jin-dal-rae Choi1,3, Sangyong Kim4 and Jaehoon Cho4 1 Department

of Chemical and Bimolecular Engineering, KAIST, Yuseong-gu, Daejeon, Republic of Korea Advanced Institute of Technology, Suwon, Republic of Korea 3 GS Caltex Corporation, Yuseong-gu, Daejeon, Republic of Korea 4 Korea Institute of Industrial Technology (KITECH), Seobuk-gu, Cheonan, Republic of Korea 2 Samsung

Volatile fatty acids (VFAs) derived from organic waste, were used as a low cost carbon source for high bioreactor productivity and titer. A multi-stage continuous high cell density culture (MSCHCDC) process was employed for economic assessment of microbial lipids for biodiesel production. In a simulation study we used a lipid yield of 0.3 g/g-VFAs, cell mass yield of 0.5 g/g-glucose or wood hydrolyzates, and employed process variables including lipid contents from 10–90% of cell mass, bioreactor productivity of 0.5–48 g/L/h, and plant capacity of 20 000–1 000 000 metric ton (MT)/year. A production cost of USD 1.048/kg-lipid was predicted with raw material costs of USD 0.2/kg for wood hydrolyzates and USD 0.15/kg for VFAs; 9 g/L/h bioreactor productivity; 100,000 MT/year production capacity; and 75% lipids content. The variables having the highest impact on microbial lipid production costs were the cost of VFAs and lipid yield, followed by lipid content, fermenter cost, and lipid productivity. The cost of raw materials accounted for 66.25% of total operating costs. This study shows that biodiesel from microbial lipids has the potential to become competitive with diesels from other sources.

Received Revised Accepted Accepted article online

23 APR 2014 21 AUG 2014 25 SEP 2014 29 SEP 2014

Supporting information available online

Keywords: Economic assessment · Low cost biomass · Microbial lipids · MSC-HCDC · VFAs

1 Introduction Biodiesel is one of the most promising alternatives to fossil fuels as its production is non-toxic, sustainable, and Correspondence: Prof. Ho Nam Chang, Laboratory of Biochemical Engineering, Department of Chemical and Biomolecular Engineering, KAIST, Yuseong-gu, Daejeon 305–701, Korea E-mail: [email protected] Current address: Qiang Fei, National Bioenergy Center, National Renewable Energy Laboratory, Golden, Colorado, U.S.A. Abbreviations: AOC, annual operating costs; B, bleed ratio; CX, concentration of cell; CL, concentration of lipid; DFC, direct fixed capital costs; MSC-HCDC, multistage continuous high cell density culture; MT, metric ton, 1000 kg; PHB, poly-β-hydroxybutyric acid; RT, residence time; TBV, total bioreactor volume; VFAs, volatile fatty acids; WH, wood hydrolyzates

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energy efficient. The world’s consumption of biodiesel was 24 billion liters per year in 2011 according to the EIA international energy stastics (http://www.eia.gov/cfapps/ ipdbproject/IEDIndex3.cfm) and the global biodiesel market is growing rapidly. In recent years much attention has been paid to developing processes utilizing “non-edible” biomass for bioenergy production. Oleaginous microorganisms carrying microbial lipid contents in excess of 20% (w/w) have been considered as alternatives to plant oils and animal fats [1]. To date, the high cost of biodiesel from fermentation of microorganisms mainly stems from the high cost of the carbon source [2], and this remains the main obstacle to the broader commercialization of these processes.

* These authors contributed equally to this work.

© 2014 The Authors. Biotechnology Journal published by Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim. This is an open access article under the terms of the Creative Commons Attribution Non-Commercial License which permits use, distribution and reproduction in any medium, provided that the Contribution is properly cited and is not used for commercial purposes.

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Biotechnol. J. 2014, 9, 1536–1546 www.biotecvisions.com


Volatile fatty acids (VFAs), which can be produced from food waste, sludge, and a variety of biodegradable organic wastes under anaerobic conditions, are the basis for a VFAs platform [3, 4]. VFAs are a potential economical alternative carbon source for lipid accumulation by oleaginous microorganisms [5]. Although many batch or fed-batch cultures have been carried out for lipid production [6–8], bioreactor productivity was not high enough for viable commercial production. Multi-stage continuous high cell density culture (MSC-HCDC) is considered to be a fermentation technology that can lead to high productivity and titer for both extracellular and intracellular products [9, 10]. High cell density culture fermentation gives a very high productivity because of its high cell density achieved by membrane cell recycling, or a moderate cell density attained by using a packed-bed combined with gravity settling [11–14]. Fei et al. [15, 16] achieved a lipid content of 55% (w/w) using VFAs as a carbon source in two-stage cultivation. Based on this result we believe that both high lipid content and lipid productivity can be improved in a MSCHCDC system. The multi-stage culture mode could be modified to provide high lipid content by using nutrient limitation strategies [5, 17]. Also recombinant bacteria species have shown to be promising alternatives for lipid accumulation processes [8]. Analysis of the entire biofuel production process from microbial lipids will allow us to design a more efficient method of microbial lipid production and to evaluate the manufacturing costs of microbial lipids and biodiesel produced at a commercial scale [18]. Technologies and vari-

ables examined in this study are: (i) MSC-HCDC technology for intracellular products; (ii) low cost wood hydrolyzates (WH) for cell mass formation; (iii) VFAs for lipid accumulation; (iv) high bioreactor productivity; (v) and plant capacity.

2 Materials and methods 2.1 Cell mass and lipid accumulation Although many studies have been performed for production of microbial lipids employing various culture modes, and utilizing bacteria, yeasts, or microalgae, the highest lipid productivity was 0.54 (g/L/h). Chang et al. [9, 19] reported that a MSC-HCDC system could give a 10 times higher productivity in terms of extracellular products (ethanol, lactic acid, monoclonal antibody, mAb) than a fed-batch culture while the product titer remained similar to that of fed-batch cultures. A single stage HCDC system yielded a very high productivity but a lower product titer [10, 11, 20]. In MSC-HCDC systems the strategy for producing intracellular products (poly-β-hydroxybutyric acid, PHB) and microbial lipids differs from those utilized to produce extracellular products. Since the production of intracellular products is dependent on cell mass, it is common to employ a two-stage process, as it is important to obtain a high as possible cell mass in the first stage, and then to maximize lipid accumulation in the second stage by imposing nitrogen or phosphate limitation. The highest cell mass obtained in fed-batch for PHB producing Ral-

Figure 1. Schematic diagram of two-stage MSC-HCDC lipid production system The first stage is a simple CSTR reactor while the second stage is a multireactor CSTR HCDC system, simulating PFR, that enables its dilution rate to run at D = 0.2/h despite its lipid accumulation of 0.1/h. : input feed flow; : substrate 1 stream; in: input stream of cell mass reactor; out: output stream of cell mass reactor; : substrate 2 stream; -1: high cell density stream; -2: low cell density stream. S1 and S2 are 180 g of glucose and 900 g of VFAs, respectively. Flow rates of stream , ,  and -2 are 0.1 L/h. Flow rates of stream  and -1 are 0.2 L/h. Concentrations (g/L) of total cell and lipid in cell mass reactor are 45 g-cell mass/L and 0 g-lipid/L, respectively. The lipid production reactor, -1 and -2 streams have concentrations of total cell mass and lipid titer are (120,90), (180, 135) and (0,0), respectively. * The working volume of lipid production tank is 3.0 L for B = 1.0 (simple continuous), 2 L for B = 0.67 (33% cell-free broth removal), 1.5 L for B = 0.5, and 1.2 L for B = 0.4 (Supporting information, A3)

© 2014 The Authors. Biotechnology Journal published by Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

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Table 1. Process variables and specifications for the simulation studies

Assumptions

Specification

Remarks

VFAs cost (USD/kg) VFAs yield (g/g) Culture mode

0; 0.10; 015;0.20 0.5b) Two-stage MSC- HCDC

(–0.6, –0.7)a) Food waste, lignocellulose [4, 36] Continuous

Microorganism

C. albidus (yeast)

0.2/h–0.1/hc); 0.18-0.36/h [16, 37]

YX/S in R1

0.5 g/g

0.5 g/g [5]

YL/S in R2

0.3 g/g (VFAs), 0.3 g/g (glucose)

0.24, 0.27;0.30–0.86 [15, 37, 38]

Maximum cell density

300/225 (B = 0.4); 240/180 (B = 0.5)

469/352 (density = 0.85), Supporting information, A1

Lipid content

75 %

[37]

Lipid productivityd)

9 g/L/h [71.28 MT/m3/y]

Supporting information, A3

Lipid production

100,000 MT/year

Lipid kg/Biodiesel L

0.85 g/L

n-hexane

Reused 100 times for 3,000 MT

Fermenter Glycerine) Lipid from lipid extracted cell Sawdust (biomass)f)

m3

density m3]

[1400 2000 USD 0.032/kg-lipid USD 0.027/kg-lipid USD 80–150/m3 (SG:0.85-0.91)

Assumption Byproduct (credit-1) Byproduct (credit-2) Supporting A5 Biomass cost

a) The treatment of foodwaste in Korea is subsidized by local government. Quantities of approximately 4 million MT/year (800,000 dry MT) from which 400,000 MT VFAs can be made to produce more than 100,000 MT microbial biodiesel. The subsidy is equivalent to –USD 0.70/kg VFAs and assuming USD 0.10 processing cost can reduce the cost to –USD 60/kg b) Experimental values from foodwaste and lignocellulosic biomass c) The growth and lipid accumulation rates of Cryptococcus albidus cells d) Proposed lipid productivity in MSC-HCDC system e) Stoichiometry of biolipid to biodiesel (10%), the current price, USD/kg f) http://www.alibaba.com/showroom/sawdust.html; specific weight: 850–910 g/L

stonia eutropha was 164 g/L containing 121 g/L intracellular PHB obtained with a high purity oxygen supply resulting in a maximum content and productivity of 75.6% and 2.42 g/L/h [21], and later a total cell mass of 281 g/L and PHB of 232 g/L in a 60 L fermenter (82.5% and 3.14 g/L/h) [22]. Figure 1 shows a schematic diagram of a two-stage MSC-HCDC lipid production system. The first stage is designed to produce a high cell density with low lipid accumulation, and can be achieved with a low carbon/ nitrogen (C/N) ratio substrate in the culture medium. A higher dilution rate can be utilized to produce a cell concentration of more than 100 g/L [9, 12, 13]. Table 1 shows the overall mass balances of substrates, cell mass, and lipids. The second stage is to accumulate high levels of microbial lipids as quickly as possible. For high-productivity lipid production, a high C/N ratio medium should be used. For the lipid accumulation rate to be higher than the cell growth rate there needs to be a low bleed ratio (B) = 0.67, 0.5 or 0.4. There is no washout of the culture in the second stage since new cells are constantly supplied from the first stage. In the second stage, several CSTRs or PFR type HCDC reactor systems can be used.

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2.2 Lipid titer and productivity In any fermentation process high product titer and bioreactor productivity are the two most important goals. If product titer is low, the product solution needs to be concentrated to the desired level to allow subsequent separation steps such as centrifugation or filtration, in order to be economically efficient. A biolipid plant with low bioreactor productivity will need a high capital investment for its construction, this will increase the manufacturing cost in relation to the direct fixed cost. In the Supporting information we supply details regarding: (i) the maximum cell density; (ii) lipid accumulation in the bioreactor; (iii) MSC-HCDC system productivity; (iv) lipid cost dependency on raw material cost; (v) recycling of waste cell mass after lipid extraction biomass to VFAs; (vi) MSCHCDC fermenter cost; (vii) the simulation process; and (viii) a comparison of autotrophic (microalgae) and heterotrophic biodiesel production systems. The maximum cell density and its product PHB are 600/480 g/L (total cell mass/PHB) and those of lipids are 469/351 (total cell mass/lipids) (Supporting information, A1). The assumptions of the PHB and lipid content of the cell were 80% and 75%, respectively. The cell density cannot be increased further due to wet volume limitations. A cell density of 200 g/L of Saccharomyces cerevisiae cells





with a 20% solid content resulted in fully packed wet cells that are too viscous for pumping [11]. Using the results given in Supporting information, A2 and A3, a bioreactor productivity of 9 g/L/h was used in this study. The bioreactor productivity can be estimated using either of the following equations: Productivity = system dilution rate × final product titer

(1)

or Productivity = final production rate (g-lipid/h)/working volume

(2)

If the final lipid concentration is 180 g/L (75% content), the total cell mass will be 240 g/L (60 g/L-cell mass, 180 g/Llipid). The dimensionless residence time (RT) required to achieve 95% accumulation for the cell mass reactor (reactor 1) is 1 and for the lipid accumulation reactor is 2.99 (PFR). Since the cell growth rate is 0.2/h and the lipid accumulation rate is 0.1/h, the lipid accumulation reactor will need a 10 h real RT unless a cell recycling method is used. Thus the strategies proposed are as follows: (i) increase the cell mass to as high a level as possible and reduce the liquid volume as low as possible; and (ii) increase cell growth and lipid production rates as high as possible. A second approach is also possible, but it may take some time to realize the goal. With the first approach 360 g/270 g will be 120 g /90 g with 3 L total volume, 180 g/135 g with 2 L, 240 g/180 g with 1.5 L and 300 g /225 g (23% free liquids 77% wet cells) with 1.2 L. All of these values are lower than 469 g/351 g (Supporting information, A1). The resulting values for lipid productivity will be 9 g/L/h for 2 L reactor volume, 10.8 g/L/h for 1.5 L, and 12.3 g/L/h for 1.2 L (Supporting information, A3). Glucose and VFAs have been demonstrated to be useful carbon substrates for the production of a high concentration of PHB and lipids in two-stage fed-batch and continuous cultures [15, 21]. It is believed that higher lipid content in cells (80%, w/w) can be obtained with VFAs as the sole carbon source in the two-stage culture system using improved microbial cells. All cost estimates (fixed capital costs and operating costs) were calculated following SuperPro designer 9.0 (SPD, Intelligen Inc., USA).

2.3 Simulation models of microbial lipid production during fermentation 2.3.1 Carbon source and other chemical components VFAs were chosen as the carbon source in the two-stage continuous HCDC process. The VFAs from Korean food waste may be –USD 0.70/kg owing to the government subsidy although the maximum quantity is limited to 4 000 000 MT wet (800 000 MT dry) per year. The US

Department of Energy (2012) estimates a biomass cost USD 30/MT from “biomass multi-years program plan”. If the conversion efficiency of biomass to VFAs is 50%, 500 kg VFAs would be obtained Then the VFAs cost would be USD 60/MT or USD 0.06/kg. USD 0.10/kg was used taking into consideration some processing costs. In addition to the VFAs, the cost of other major raw materials are (USD/MT): NH3, 200; n-Hexane, 1500; water, 0.305. The cost of other chemical components, such as inorganic salts, was not included because their costs were an insignificant contribution to the final production cost of microbial lipids. Table 2 shows that the conversion of lipid/kg to biodiesel/L is 0.85; the carbon source in reactor 1 is WH; the VFAs were used in reactor 2. Lipid productivity is assumed to be 9/L/h and 3 000 MT of hexane used for lipid extraction. Table 3 shows major equipment and purchase costs for the production of 100 000 MT lipids. All equipment sizes are dependent on the fermenter size and based on the bioreactor productivity.

2.3.2 Fermentation and process description This process is divided into four sections: (i) seed and medium preparation; (ii) fermentation; (iii) cell mass washing and recovery; and (iv) lipid extraction. The Soxhlet extractor method is used in the lipid extraction process, extracting the lipids from dried cell mass [6]. The feeding solution to reactor 2 is composed of VFAs, a mixture of acetic acid, propionic acid, and butyric acid at a ratio of 5:1:5 with no additional nitrogen source [4].

2.3.3 Stoichiometry of raw materials to make biodiesel Figure 2 shows that the production cost of microbial lipid is a function of lipid content of the cells, the raw materials cost, and stoichiometry. The details of the raw material cost calculations are given in Supporting information, A4 and Fig. S2B. During cell growth periods with 100% substrate conversion, no lipid formation is assumed although cells synthesize essential lipids for cell growth such as cell membranes [22]. Intracellular lipid is formed as a result of cell inoculation and fermentation, and cells and lipids are recovered by centrifugation and solvent extraction. Soybean has a density of 0.849 to 0.892 while palm oil has a density of 0.809 to 0.892 kg/L depending on the grade [23]. A final conversion cost of USD 0.06 USD/L from lipid to diesel is added [24].

2.3.4 Plant capacity and capital investment The economic assessment was considered with the assumptions listed in Table 2. For the production of 100 000 MT lipid in this two-stage continuous HCDC process, a working volume of 1403 m3 was needed and the fermenter volume for MSC-HCDC bioreactor was 2000 m3 (70% working volume), with 71.28 MT lipid/(m3 year). The capital costs reported in this study were based on SPD 9.0


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Table 2. Major equipment specifications and purchase costs for a 100 000 MT lipids/year

Processing step

Equipment

Size

Medium preparation

Peristaltic pump Heat sterilizer

Power = 1.85, 3.70 kW Throughput = 46.72, 93.16 m3/h

Fermentation

Compressor

Pressure change = 7 bar Capacity = 59 m3/h Throughput = 0.11, 0.56, 1.11 m3/s Throughput = 0.85, 2.84, 2.84 m3/s Volume = 20 m3 Volume = 2000 m3 Bleed = 0.67

Air filter (inlet) Air filter (emission) Seed fermenter MSC-HCDCb) Pack-bed Cell washing and recovery Lipid Extraction

Disk-stack centrifuge Spray dryer Peristaltic pump Blending tank Disk-stack centrifuge Homogenizer Condenser Distillation column

Through put = 104.95 m3/h Diameter = 4.66 m Length = 13.99 m Power = 0.02 kW Volume = 109.35 m3 Through put = 77.07 m3/h Through put = 37.55 m3/h Area = 72.17 m2 Height = 5.60 m Diameter = 1.30 m

Total equipment Cost of unlisted equipment (20% of total equipment purchase cost) Total

Quantity

Costa) (× 103 USD)

2 2

19 1 815

15

675

3 7 3 1 1

40 615 300 1100 99

1

901

1

332

1 1 1 2 1

3 509 114 300 37

1

39 6 898 1 725 8 623

a) Equipment cost is based on SuperPro designer and http://www.alibaba.com b) Cost of MSC-HCDC described in Supporting information, A6.

Figure 2. Production cost of microbial lipids. (A) Effect of lipid content on lipid production cost. (B) Various raw materials and recycling (R) of waste cell mass to VFAs with lipid content , 75% and cell mass, 25%. Cell yield = 0.5 kg/kg for glucose (G), wood hydrolyzates (WH) and VFAs. Lipid yield is 0.3 kglipid/kg of G and WH, 0.4 kg-lipid/kg VFAs. SN-a: cell mass for G and lipid formation for G; SN-b: G, WH; SN-c: G, VFAs; SN-d: WH, VFAs (R = 0%); SN-e: WH, VFAs (R = 50%); SN-f: WH, VFAs (R = 100%). White bar and dark gray bar indicate the carbon source cost for cell mass and lipid formation, respectively.

and the alibaba.com web site (http://www.alibaba.com). Since there is little or no information on costs of continuous culturing, it is assumed that the capital and operating

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costs of MSC-HCDC and fed-batch are similar to each other on a capacity basis while the cost of MSC-HCDC is based on Supporting information, A6. A list of specifica-





Table 3. Relative magnitude of equipment costs for a 100,000 MT microbial lipids/year fermentation facility

Process operation Medium preparation Fermentation Cell mass washing and recovery Lipid extraction Total

% total cost 26.6 41.0 17.9 14.5 100.0

tions and purchase costs of major equipment are given in Table 3.

2.4 Cost of biodiesel production The economic assessment of biodiesel production from VFAs based lipids was estimated based on the Haas’ process, which simulated a process model for production of 10 000 000 gallons per year biodiesel [25].

3 Results 3.1 Microbial lipids produced at a higher productivity 3.1.1 Mass balance of cell mass and biolipids Figure 1 shows the overall mass balances in a MSC-HCDC lipid production system where 180 g glucose is supplied via stream  to yield a 90 g cell mass delivered for lipid production via tank , and 270 g lipid is produced using 900 gVFAs from stream . The total cell mass concentration is 360 g (lipid content, 75%) and is discharged to the final cell mass separation units designated cell rich  -1 and cell free  -2. Nearly all the cells are recovered via stream  -1 while  -2 stream is cell free. Hollow fiber can produce B = 0 resulting in total cell recycling [11]. In the up-flow packed-bed cell recycle system [14] B = 0 is not possible but can be kept as high as B = 0.67, 0.5, or 0.4. Cell and lipid titers are obtained by dividing those values with their respective flow rates. The concentrations of total cell mass and lipid should not exceed 469 g/L and 351 g/L that are the maximum theoretical values. The total cell mass concentration, C × (g/L), and lipid concentration, CL (g/L), are 180 and 135 g/L, respectively, at B = 0.67. These values are lower than the C × = 281 and CL 232 g/L obtained by Ryu et al. [25] in their PHB experiment. Stream  is dependent on how much water is removed, by centrifugation or the use of membranes, and this value needs to be determined more accurately by experimental methods. The Supporting information, A1 estimates 60% (120 g cell mass, 480 g PHB) to be the maximum yield of PHB, while for lipid 50% will be the maximum yield (100 g cell mass, 400 g lipid). 180 g / 135 g is 45 g cell mass and 135 g lipid, which indicates that can be

gains can be made from increases in cell density. Again for convenience 100% lipid recovery by extraction is assumed.

3.1.2 MSC-HCDC for a high productivity and titer The second stage lipid accumulation process employs a MSC-HCDC bioreactor system having cell recycling with an up-flow packed bed, a higher dilution rate with 0.2/h instead of 0.1/h as was used in the simulation. This is essential since employing a hollow-fiber system would be troublesome due to plugging, fouling, and additional costs, but the bleed rate of B = 0.5 or 0.4 can be achieved in an up-flow packed bed cell recycle system to obtain two-fold higher lipid accumulation rates per liter. A separation of cell retention time from its hydraulic retention time makes it possible to use higher dilution rates without washouts. Tables 1–5 estimate the capital and operating costs for the production of microbial lipids produced using WH and VFAs as the carbon source. After fermentation the entire cell washing and extraction operation could be continuously and simultaneously carried out. There are several possible methods for the extraction of lipid from cell mass. Among these methods, the Soxhlet extractor method (washing with n-hexane and then evaporating the mixed solution) allows high efficiency lipid extraction from dried cell mass [17]. Furthermore, the n-hexane solvent can be recovered by a condensation process to a high purity, and can therefore be reused resulting in a more economical and environmentally friendly process.

3.1.3 Contribution of carbon sources to cell mass and biolipids production costs Figure 2A shows raw materials cost per kg of lipid from WH for the first stage (cell mass formation) and VFAs for the second stage (lipid formation) by varying lipid content from 100% to 10%. L100 (100% lipid content) yields USD 0.500; L075 USD 0.633; and L010 USD 4.100. Decreased lipid content of the cell from 0.8 to 0.1 will increase lipid cost from USD 0.600 to USD 4.100. A lower lipid content than 1.0 will result in additional costs of increased cell mass production. Figure 2B shows the raw material cost of 25% cell mass and 75% lipid content: SN-a for G (glucose)/G USD 1.600; SN-b for G/WH USD 0.933; SN-c for G/VFAs USD 0.767; SN-d USD 0.633; SN-e USD 0.619; SN-f USD 0.606. As expected, glucose substrates for cell mass and lipid accumulation are the most expensive.

3.1.4 Capital costs The total equipment cost was estimated to be USD 8 623 000. Table 2A shows the proportional contribution to the total equipment costs from each of the major process operations. Fermentation equipment accounts for 41.0% of the total equipment costs (Table 3). These equipment costs were calculated through classical chemical engineering cost estimation techniques


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Table 4. Total capital investment for a 100 000 MT microbial lipid

Fixed capital estimate summary

Cost (thousand USD)

Total plant direct cost (TPDC) (physical cost) 1. Equipment purchase cost (PC) 2. Installation (45.6% PC) 3. Process piping (35% PC) 4. Instrumentation (40% PC) 5. Insulation (3% PC) 6. Electrical (10% PC) 7. Buildings (45% PC) 8. Yard improvement (15% PC) 9. Auxiliary facilities (40% PC) TPDC Total

8 623 3 932 3 018 3 449 259 862 3 880 1 293 3 449 28 766

Total plant indirect cost (TPIC) 1. Engineering (25% TPDC) 2. Construction (35% TPDC) TPIC Total

7 191 10 068 17 259

Other cost (OTC) 1. Contractor’s fee (5% ( TPDC + TPIC)) 2. Contingency (10% ( TPDC + TPIC)) OTC Total Total fixed capital (DFC) TPDC + TPIC + OTC

2 301 4 602 6 904 52 929

[26]. Total capital cost estimates include direct fixed capital costs (DFC), indirect fixed capital costs, and other cost (OTC), the details of which are summarized in Table 4. The total capital investment for the process with a production of 100 000 MT microbial lipids/year was calculated to be USD 52 929 000, while the total direct fixed capital investment would be USD 28 766 000, which accounts for 54.3% of total capital costs.

3.1.5 Operating costs Table 5 and Table 6 show annual operating costs (AOC) in terms of raw materials, utilities, labor dependent, administration and overhead expenses, and direct fixed cost dependent items. The total AOC amounted to USD 104 764 000. The facility dependent items are estimated to be approximately USD 9 952 k, contributing 9.50% of the AOC. The cost of utilities, labor-dependent items, and waste treatment are calculated to be USD 22 457 000, USD 1 172 000 and USD 1 774 000, respectively. The raw materials accounted for 66.25% of AOC (Table 6), indicating that the cost of raw materials is the main component of the AOC of microbial lipid production. In the breakdown of the annual raw material cost (USD 69 409 000), we find that 91.25% of the raw materials cost is the cost of the carbon sources (WH and VFAs). The amount of n-hexane for

Table 5. Total operating costs for a 100,000 MT of microbial lipids plant

Operating costs estimate summary

Amount/year

Unit cost a)

Raw materials 1-1. Carbon source (VFAs) 1-2. Wood hydrolyzates 2. NH3 3. Water 4. n-Hexanea) Subtotal

333 333 MT 66 666 MT 6 500 MT 904 166 MT 3 000 MT

USD 150/MT USD 200/MT USD 200/MT USD 0.305/MT USD 1500/MT

50 000 13 333 1 300 276 4 500 69 409

Utilities 1. Electricityb) 2. Cooling water 3. Chilled water 4. Steam Subtotal

1.33 × 108 kWh 1.57 × 107 MT 1.59 × 107 MT 497 422 MT

USD 0.07/kWh USD 0.05/MT USD 0.40/MT USD 12.00/MT

9 309 817 6 361 5 969 22 457

117 216 h

USD 10/h

1 172

942 563 MT 731 362 MT

USD 0.33/MT USD 2/kg

311 1 463 1 774

Labor 1. Operating labor Waste treatment 1. Water waste 2. Organic waste Subtotal Facility-dependent 1. Depreciation 2. Other facility-dependent Subtotal

Cost (× 103 USD)

5 028 4 924 9 952

Total operating cost

104 764

a) http:// www.alibaba.com b) US average industrial electricity cost, (EIA, 2014) http://www.eia.gov/electricity/monthly/epm_table_grapher.cfm?t=epmt_5_6_a

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Table 6. Annual operating costs for a 100 000 MT microbial lipids plant

Description Raw materials Carbon Source-WH + VFAs Utilities Labor-dependent Facility- dependent Waste treatment Subtotal operating costs Glycerin Empty cell mass to VFAsa) Biodiesel cost (USD /L)c) Conversion cost of lipid to biodiesel Net cost of biodiesel (USD /L)

Cost (× 103 USD)

% of AOC

69 409 63 333 22 457 1 172 9 952 1 774 104 764

66.25 60.45 21.44 1.12 9.50 1.69 100.0

Unit cost (USD/kg) 0.694 0.633 0.225 0.018 0.100 0.018 1.048 –0.032 –0.027b) 0.840 0.060 0.901

a) VFAs yield from lipid extracted cell mass = 0.27 g/g. (Supporting information, A5) b) Lipid price from lipid extracted cell mass was set as USD 1/L. c) Lipid (USD/kg)*0.85 = Biodiesel (USD/L)

extracting 40 000 MT lipids is estimated to be 1 200 MT, meaning that this amount of n-hexane was assumed to be used and re-used 85 times. To extract 100 000 MT of lipids, the total amount of n-hexane will be 3 000 MT. If the cost of the carbon source increases, the total raw material costs will increase dramatically. On the basis of operating labor, estimated at 7920 h/year, the total labordependent costs amount to 1.12% of AOC. 21.44% of AOC was estimated for utilities costs, which was calculated based on electricity, cooling water, chilled water, and steam. The unit cost per kg lipid is USD 1.048 of which raw material cost is USD 0.694 (USD 0.633 carbon source) and the remaining USD 0.354 is the processing cost. Also, if 50% of the cell mass is converted to VFAs production, the raw material cost will decrease to USD 0.681 (total cost

USD 1.035) and the 100% recycling of n-hexane will reduce the raw material cost to USD 0.667 (total cost USD 1.021). The final lipid price is reduced a further USD 0.032 due to the glycerin credit. If the lipid content of the cells decreases, there is a critical value at which no favorable economic assessment is possible.

3.1.6 Effect of productivity and plant size The basic parameters are a productivity of 9 g/L/h and a plant capacity of 100 000 MT/year. Bioreactor productivity can go up to 48 g/L/h if a high cell density culture with cell recycling and fast growing E. coli cells are used. The reactor size may decrease or increase depending on productivity resulting in a concomitant change in its economics. With a fixed carbon cost of USD 0.633/kg,

Figure 3. Operating cost of microbial lipids with lipid productivity and plant size (75% lipid content, 25% cell mass, USD 0.2/kg WH for cell mass and USD 0.15/kg VFAs for lipid formation). (A) Effect of productivity. (B) Effect of plant capacity. White and dark gray bars indicate operation cost without carbon source and operation cost of carbon source, respectively.


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Table 7. Lipid production cost with various process variables.

Factor

Value

C sourcea (USD/kg)

w/o C sourcea) (USD/kg)

Lipid costb) (USD/kg)

Biodieselc) cost (USD/L)

VFAs cost (USD/ton)

0 0.10 0.15 0.20

0.133 0.466 0.633 0.800

0.415 0.415 0.415 0.415

0.548 0.881 1.048 1.215

0.526 0.809 0.951 1.093

Lipid/VFAs yield (g lipid/g VFAs)

0.20 0.27 0.3 0.4

1.133 0.689 0.633 0.508

0.415 0.415 0.415 0.415

1.548 1.104 1.048 0.923

1.376 0.998 0.951 0.845

Lipid content (%)

50 60 67.5 75

0.900 0.766 0.693 0.633

0.415 0.415 0.415 0.415

1.315 1.181 1.108 1.048

1.178 1.064 1.002 0.951

Fermenter cost (× 103 USD)

1100 3000 5000

0.633 0.633 0.633

0.415 0.441 0.469

1.048 1.074 1.102

0.951 0.973 0.997

Production capacity (× 103 ton)

50 100 500

0.633 0.633 0.633

0.583 0.415 0.280

1.216 1.048 0.913

1.094 0.951 0.836

Lipid productivity (g/L /h)

1.5 6 9

0.633 0.633 0.633

0.789 0.489 0.415

1.422 1.122 1.048

1.269 1.014 0.951

1.106

1.000

Upper limitd)

a) Operating cost b) Standard condition: VFAs cost = USD 0.15/ton, VFAs yield = 0.3 g/g, Fermenter cost = 1100 × 103 USD, Production capacity = 100,000 MT, Lipid productivity = 9 g/L/h. c) Biodiesel cost (USD/L) = Lipid cost (USD/kg) 0.85 + 0.06 d) Upper limit of biomass cost is USD 0.188–0.256/kg at VFAs yield = 0.3–0.4, lipid content = 0.75-0.8, biomass yield = 0.8

0.5g/L/h yielded a lipid cost of USD 2.324/kg and an increase in production to 48g/L/h reduced the lipid cost to USD 1.048/kg. A productivity of greater than 6 g/L/h did not significantly change the DFC (Fig. 3A). The smallest assessed production plant of 20 000 MT/ year gives USD 1.705/kg and 500 000 MT/year yields USD 0.913/kg. The lipid production cost at the largest assessed plant size of 1 000 000 MT per year is only USD 0.876/kg (Fig. 3B).

3.2 Process variables on microbial lipids-based biodiesel Table 7 summarizes the biodiesel cost assessment with various process variables. Microbial lipids based biodiesel appears to be the only form of biodiesel that has the potential to be competitive with fossil derived diesel. When VFAs based lipids are used for biodiesel production, the cost of biodiesel ranges from USD 0.526/L to USD 0.809/L at USD 0.00 to USD 0.10 of VFAs cost , which is lower than fossil diesel priced at USD 0.914/L. Because of their low cost, VFAs could be an excellent alternative carbon sources for commercial processes based on oleaginous

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microorganisms. As shown in Fig. 2B, due to its comparatively high cost glucose cannot compete with VFAs as a carbon source. In addition to glucose and VFAs, there are other low cost materials which could be used as the carbon source for lipid production, such as xylose and wastewater [27]. Providing sufficient oxygen to maintain aerobic conditions can be costly but it is necessary for large-scale industrial production [28]. When lipid is produced in HCDC, oxygen transfer rate may often act as a limiting factor[29]. The production cost of microbial lipids based biodiesel is much lower than other biodiesels produced from various feed stocks, such as soybean oil (USD 1.04/L), palm oil (USD 0.851/L) and microalgae (USD 1.4/L). The high value of these feed stocks makes it very challenging to produce a cost competitive biodiesel fuel. In the US, fossil diesel prices (excluding tax) during 2013 were generally in the range of USD 0.88-0.98 (Energy information administration http://www.eia.gov/countries/prices/ dieselextax.cfm), a price considerably lower than the cost of biodiesel production derived from vegetable oils. Although the cost of microbial lipid biodiesel determined





in this study may be a reference for future studies there is still room for further improvement of cells by metabolic engineering [30] as well as in the area of process technology. The future of microbial biofuel production may be brighter than microalgae-based biodiesels or plant oils that rely on area-based cultivation in tropical, subtropical and temperate climate regions, as VFAs derived from biomass can be obtained anywhere in the world if good storage technology becomes available. Chisti claims that biodiesel from microalgae should be less than USD 0.759/L at an oil price of USD 110/barrel [31]. The manufacturing price of microbial diesel is slightly less than this value. Currently the US government subsidy for biodiesel is USD 0.29 per energy equivalent liter (EEL) [32]. Biodiesel from microbial lipids may be competitive even without this subsidy.

4 Discussion The current investigation used an oleaginous yeast for lipid accumulation, but other microorganisms such as fungi have also been employed [33, 34]. Microbial production of microbial lipid was economically assessed using VFAs from low cost biomass, and MSC-HCDC with a high productivity of 9 g/L/h. The predicted cost was USD 1.048/kg or USD 0.951/L (including credit, it is USD 0.901/L) at a plant capacity of 100 000 MT/year. Since the above value is based on 95% of the maximum accumulation, it needs to be modified for 100% conversion. In this case there would be an approximately 3% cost increase from 95% lipid accumulation basis, but it depends on RT in the lipid production reactor. If bleed ratio (B) = 1 (no cell recycle), the lipid accumulation is 95% where RT = 10 h, 0.1/h and total bioreactor volume (TBV) is 4 L (= 1 + 3); B = 0.667, 98.9%, RT = 3.0/0.2 = 15 h, TVB = 3 L; B = 0.5, 99.75% at RT = 20 h, TBV = 2.5 L; B = 0.4, 99.94% at RT = 25 h, TBV = 2.2 L. Longer RT is assured with a fixed reactor volume as it is for various cell recycling operations compared to no cell recycling. This is the advantage of the cell recycle system (Supporting information, A3). The current biodiesel production model using low cost VFAs can compete with palm oil (USD 0.851/L), soybean oil (USD 1.044/L), microalgae oil (USD 1.40/L) or petroleum diesel (USD 0.914-1.039/L). The comparison remains favorable when also factoring in transportation costs between the site of biodiesel production and consumption, as well as major improvements in the lipid yield (Table 1). The lipid yield and lipid productivity can be improved further by selecting or modifying existing microbial strains in combination with increasing bioprocessing efficiency, which will substantially reduce production costs to a value lower than USD 1.00/kg-lipid. In addition advances in synthetic biology will be help reduce the cost of lipid separation from microbial cells [35].

Microbial biodiesel can be produced anywhere in the world using indigenous biomass while plant or microalgae oils can only be produced in tropical or subtropical countries. The most competitive bioprocess for microbial biodiesel can use more expensive raw materials than other processes. Biomass can be abundant regardless of geographic location and climate, and the ability to set up microbial biofuel production systems anywhere minimizes logistic problems of transporting bulk biomass to different locations. In order to become the world’s best biofuel raw material microbial VFAs yields need to be improved above 0.5 g VFAs/g-biomass. This is probably quite feasible since nearly all the components of biomass (carbohydrate, fats and lipids, proteins) including lignin can be a source of VFAs. Furthermore the lipid yield of oleaginous microbes should be improved to over 0.3 g/g VFAs. In addition biomass production needs to be considered from the viewpoint of land cost, biomass productivity, and VFAs yields, etc. (Supporting information, A8). The VFAs from Korean food waste may cost –USD 0.60/kg by adding a USD 0.10/kg processing cost to –USD 70/kg. This large credit can be used to cover raw materials costing more than USD 0.15/kg for extended production capacities or making a smaller plant more economically feasible.

The authors would like to thank Dr. Christopher J. Brigham and John W. Quimby for their discussions, and editing of the manuscript. This work was supported by the National Research Foundation of Korea Grant (NRF–2011–0009582). The authors declare no commercial or financial conflict of interest.

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ISSN 1860-6768 · BJIOAM 9 (12) 1459–1623 (2014) · Vol. 9 · December 2014

Systems & Synthetic Biology · Nanobiotech · Medicine

12/2014 Asian Congress of Biotechnology 2013


This Special issue covers contributions from the Asian Congress of Biotechnology 2013 (New Delhi, India, December 2013) in collaboration with the Asian Federation of Biotechnology (AFOB). The issue is edited by Virendra Bisaria and Akihiko Kondo and includes articles on stem cells, algae biotechnology and recombinant protein. The cover image shows the cell-penetrating mechanism of the 30Kc19 protein derived from the silkworm hemolymph. The silkworm image also symbolizes the Asian region. Image designed by Helpdesign. See the article by Park et al. http://dx.doi.org/10.1002/biot.201400253

Biotechnology Journal – list of articles published in the December 2014 issue. Editorial: Asian Congress on Biotechnology 2013 Akihiko Kondo and Virendra S. Bisaria http://dx.doi.org/10.1002/biot.201400774 Editorial: Building on the BTJ experience to foster scientific and research communication Judy Peng http://dx.doi.org/10.1002/biot.201400785 Commentary The passive strategy: Increasing the force in the battle against influenza Reingard Grabherr

http://dx.doi.org/10.1002/biot.201400409 Commentary Production of vitamin B12 in recombinant Escherichia coli: An important step for heterologous production of structurally complex small molecules Yin Li

http://dx.doi.org/10.1002/biot.201400472 Review Expanding frontiers in plant transcriptomics in aid of functional genomics and molecular breeding Pinky Agarwal, Swarup K. Parida, Arunima Mahto, Sweta Das, Iny Elizebeth Mathew, Naveen Malik and Akhilesh K. Tyagi

http://dx.doi.org/10.1002/biot.201400063 Review Aquatic proteins with repetitive motifs provide insights to bioengineering of novel biomaterials Yun Jung Yang, Dooyup Jung, Byeongseon Yang, Byeong Hee Hwang and Hyung Joon Cha

http://dx.doi.org/10.1002/biot.201400070 Review Open and continuous fermentation: Products, conditions and bioprocess economy Teng Li, Xiang-bin Chen, Jin-chun Chen, Qiong Wu and Guo-Qiang Chen

http://dx.doi.org/10.1002/biot.201400084

Technical Report Raman spectroscopy provides a rapid, non-invasive method for quantitation of starch in live, unicellular microalgae Yuetong Ji, Yuehui He, Yanbin Cui, Tingting Wang, Yun Wang, Yuanguang Li, Wei E. Huang, and Jian Xu

http://dx.doi.org/10.1002/biot.201400165 Research Article Zinc, magnesium, and calcium ion supplementation confers tolerance to acetic acid stress in industrial Saccharomyces cerevisiae utilizing xylose Ku Syahidah Ku Ismail, Takatoshi Sakamoto, Tomohisa Hasunuma, Xin-Qing Zhao and Akihiko Kondo

http://dx.doi.org/10.1002/biot.201300553 Research Article Coenzyme B12 can be produced by engineered Escherichia coli under both anaerobic and aerobic conditions Yeounjoo Ko, Somasundar Ashok, Satish Kumar Ainala, Mugesh Sankaranarayanan, Ah Yeong Chun, Gyoo Yeol Jung and Sunghoon Park

http://dx.doi.org/10.1002/biot.201400221 Research Article Volatile fatty acids derived from waste organics provide an economical carbon source for microbial lipids/biodiesel production Gwon Woo Park, Qiang Fei, Kwonsu Jung, Ho Nam Chang, Yeu-Chun Kim, Nag-jong Kim, Jin-dal-rae Choi, Sangyong Kim and Jaehoon Cho

http://dx.doi.org/10.1002/biot.201400266 Research Article Photon up-conversion increases biomass yield in Chlorella vulgaris Kavya R. Menon, Steffi Jose and Gadi K. Suraishkumar

http://dx.doi.org/10.1002/biot.201400216 Research Article Chromosomal integration of hyaluronic acid synthesis (has) genes enhances the molecular weight of hyaluronan produced in Lactococcus lactis Rothangmawi Victoria Hmar, Shashi Bala Prasad, Guhan Jayaraman and Kadathur B. Ramachandran

http://dx.doi.org/10.1002/biot.201400215 © 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim


Research Article Ionic liquids as novel solvents for the synthesis of sugar fatty acid ester

Research Article Humanized antibody neutralizing 2009 pandemic H1N1 virus

Ngoc Lan Mai, Kihun Ahn, Sang Woo Bae, Dong Woo Shin, Vivek Kumar Morya and Yoon-Mo Koo

Nachiket Shembekar, Vamsee V Aditya Mallajosyula, Piyush Chaudhary, Vaibhav Upadhyay, Raghavan Varadarajan and Satish Kumar Gupta



Research Article Electron donation to an archaeal cytochrome P450 is enhanced by PCNA-mediated selective complex formation with foreign redox proteins

Research Article Efficient myogenic commitment of human mesenchymal stem cells on biomimetic materials replicating myoblast topography

Risa Suzuki, Hidehiko Hirakawa and Teruyuki Nagamune

Eunjee A. Lee, Sung-Gap Im and Nathaniel S. Hwang



Research Article Dimerization of 30Kc19 protein in the presence of amphiphilic moiety and importance of Cys-57 during cell penetration

Research Article Modulation of the stemness and osteogenic differentiation of human mesenchymal stem cells by controlling RGD concentrations of poly(carboxybetaine) hydrogel

Hee Ho Park, Youngsoo Sohn, Ji Woo Yeo, Ju Hyun Park, Hong Jai Lee, Jina Ryu, Won Jong Rhee and Tai Hyun Park


Hsiu-Wen Chien, Szu-Wei Fu, Ai-Yun Shih and Wei-Bor Tsai


© 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim


Progress and Challenges in Microalgal Biodiesel Production.

Biodiesel Production from Chlorella protothecoides Oil by Microwave-Assisted Transesterification.

Mixotrophic cultivation of microalgae for biodiesel production: status and prospects.

Production of bioethanol and biodiesel using instant noodle waste.

Biodiesel production from indigenous microalgae grown in wastewater.

Identification of critical parameters in liquid enzyme-catalyzed biodiesel production.

Production of biodiesel from carbon sources of macroalgae, Laminaria japonica.

Mechanistic modeling of biodiesel production using a liquid lipase formulation.

Ultrasound-assisted biodiesel production from Camelina sativa oil.

Isolation and characterization of microalgae for biodiesel production from seawater.

A Review of Microwave-Assisted Reactions for Biodiesel Production.

Heterogeneous catalysis for sustainable biodiesel production via esterification and transesterification.

Reducing electrocoagulation harvesting costs for practical microalgal biodiesel production.

Highly efficient enzymatic biodiesel production promoted by particle-induced emulsification.

Efficient harvesting of Chaetoceros calcitrans for biodiesel production.

Assessing the greenhouse gas emissions of Brazilian soybean biodiesel production.

Biodiesel production from yeast Cryptococcus sp. using Jerusalem artichoke.

Lipase-catalyzed process for biodiesel production: protein engineering and lipase production.

Enhancement of lipid production and fatty acid profiling in Chlamydomonas reinhardtii, CC1010 for biodiesel production.

In vitro Fermentation, Digestion Kinetics and Methane Production of Oilseed Press Cakes from Biodiesel Production.

A multi-criteria analysis approach for ranking and selection of microorganisms for the production of oils for biodiesel production.

Biodiesel production from Nannochloropsis gaditana lipids through transesterification catalyzed by Rhizopus oryzae lipase.

Selection of microalgae for biodiesel production in a scalable outdoor photobioreactor in north China.

Characterization of Amphora sp., a newly isolated diatom wild strain, potentially usable for biodiesel production.